The present invention relates to novel derivatives of nucleosides and their use for the synthesis of oligonucleotides.
It more specifically relates to derivatives of nucleosides formed from pyrimidine or purine bases having exocyclic NH.sub.2 groups, i.e. nucleosides formed from adenine, guanine or cytosine, which can in particular be used for the synthesis of oligonucleotides.
The synthesis of oligonucleotides consists of linking together the nucleosides by a phosphate group to form an DNA (deoxyribonucleic acid) chain or RNA (ribonucleic acid) chain. In this bond, the internucleotide phosphate groups still link the hydroxyl function in the 3' position of a nucleoside with the hydroxyl function in the 5' position of another nucleoside. Thus, during the synthesis reaction only the 3' and 5' ends of the nucleosides are subject to an action and the nucleic base (purine or pyrimidine) used must not be involved during said bonding.
When these bases comprise exocyclic NH.sub.2 groups, it is necessary to protect these groups during the synthesis of the oligonucleotides, because they are too reactive and may interfere with the synthesis reactions.
This protection of exocyclic NH.sub.2 groups must satisfy the following : it must be selective and easy to carry out, it must not induce reactivity modifications to the other nucleoside sites and must be stable throughout the oligonucleotide synthesis stages and it must be eliminatable under gentle conditions without destroying the oligonucleotide which has been synthesized.
The exocyclic NH.sub.2 groups of nucleosides have most frequently been protected in the form of amides, e.g. by means of benzoyl or anisoyl groups in the case of adenine and cytosine, as described by H. SCHALLER et al in J. Amer. Chem. Soc, 1963, vol 85, pp 3821-3827 and by means of the isobutyryl group in the case of guanine, as described by H. BUCHI and H. KHORANA in J. Mol. Biol, 1972, vol 72, pp 251-288.
These protective groups can be eliminated at the end of synthesis by the action of 28% ammonia for 17 hours and at a temperature of 60.degree. C., as has been recommended. However, the NMR of the proton shows that under these conditions all the isobutyryl groups of the guanine are not eliminated. It is therefore preferable to raise the reaction times to 72 hours, but still at a temperature of 60.degree. C.
This procedure for eliminating protective groups constitutes a disadvantage, because the conditions used are not sufficiently gentle to permit use with modified nucleosides which are not very stable in the alkaline medium, as is e.g. the case with 5,6-dihydrothymidine.
Research has also been carried out on the possibility of using other acyl groups which are easier to eliminate, more particularly usable for the synthesis of oligonucleotides from unstable nucleosides by the methodology of synthesis on a support, which consists of fixing the first nucleoside of the chain to a support, generally of silica and then successively carrying out condensation cycles for fixing the other nucleosides in the desired order to the first nucleoside. The use of easier to eliminate acyl groups makes it possible to obtain a better deprotection yield. This point is very important, because the presence of incompletely deprotected bases constitutes a disadvantage for the use of the products obtained.
The present invention relates to novel derivatives of nucleosides having protective groups of the acyl type which can be easily eliminated.